1. Field of the Invention
This invention relates to an immunoassay of an analyte and materials used therein, and more particularly relates to a method and materials for an immunoassay using a novel alkaline phosphatase substrate composition.
2. Background of the Invention
Assay systems which are both rapid and sensitive have been developed to determine the concentration of a substance in a fluid. Immunoassays depend on the binding of an antigen or hapten to a specific antibody and have been particularly useful because they give high levels of specificity and sensitivity. These assays generally employ one of the above reaqents in labeled form, the labeled reagent often being referred to as the tracer. Immunoassay procedures may be carried out in solution or on a solid support and may be either heterogeneous, requiring a separation of bound tracer from free (unbound) tracer or homogeneous in which a separation step is not required.
Enzymes have often been used as labels in immunoassay. In conventional enzyme immunoassay (EIA), an enzyme is covalently conjugated with one component of a specifically binding antigen-antibody pair, and the resulting enzyme conjugate is reacted with a substrate to produce a signal which is detected and measured. The signal may be a color change, detected with the naked eye or by a spectrophotometric technique, or may be conversion of the substrate to a product detected by fluorescence.
Enzymes to be used in an immunoassay must be stable, highly reactive, available in a highly puried form, yield stable conjugates (tracers) and be inexpensive, safe and convenient to use. An enzyme which meets these criteria and is often used in immunoassay is alkaline phosphatase (AP).
AP occurs in substantially all animal species, and, in mammals, occurs in two forms. One form is widely distributed in a variety of tissues while the other is confined to the intestine.
AP catalyzes the cleavage of phosphate groups from generally colorless phosphorylated substrates to give colored products. Since the enzyme is active at alkaline pH, assay systems using AP generally include a buffer which maintains the pH of the assay medium in the range 7-9.
Levamisole, (-)2,3,5,6-tetrahydro-6-phenylimi-dazo[1,2-b]thiazole, is well-known to be a potent inhibitor of AP from several mammalian tissue types. It is, however, approximately 100-fold less potent toward AP from the intestine (Van Belle, Biochim. et Biophys. Acta., 289, 158 (1972).
Advantage has been taken of this selectivity in immunoassay. When used in an assay, levamisole does not interfere with the specific immuno signal generated by calf intestinal AP but does reduce the nonspecific signal which arises from any nonintestinal AP which may be present in a clinical sample. Morris et al., in the Journal of Immunological Methods, 68, 11 (1984) discloses detection of the binding of monoclonal antibodies to antigens on the surface of whole cells with a conjugate of calf intestinal AP and goat anti-mouse Igb in the presence of a substrate and levamisole added to inhibit AP of nonintestinal origin.
Ponder et al. in the Journal of Histochemistry and Cytochemistry, 29, 981 (1981), discloses detection of mouse H2 antigen in tissue slices by incubating the tissue slices with anti-mouse H2 antibody and treating with an intestinal AP labeled conjugate followed by a substrate for the enzyme and levamisole to inhibit nonintestinal AP. In the Ponder et al. method, levamisole at a concentration of 1 mM is added to a filtered AP substrate solution prior to combining the substrate with the tissue slices.
Levamisole, while useful in EIA as disclosed in the above patents, is known to be unstable at alkaline pH. Dickinson et al., in Analyst, 96, 248 (1971), have shown that levamisole is rapidly decomposed at elevated temperature, and that at pH 7.9, it is decomposed 70 times faster than at pH 2. Accordingly, up until the present invention, levamisole and the high pH assay buffer required for AP activity could not be premixed prior to performing the assay. Significant advantages in time, cost and assay convenience which accrue from premixing assay reagents thus could not be achieved. The present invention is directed to overcoming this deficiency in prior art assays.